Detection of carbapenemase producing Acinetobacter baumannii ST19 from Georgia and Ukraine carrying bla OXA-23, bla OXA-72, and/or bla NDM-5, December 2019 to June 2023

In 2003−2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23, bla OXA-72, and/or bla NDM-5. Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.


Acinetobacter baumannii ST19 collection and characteristics
From 5,436 A. baumannii genomes of MDR strains (as defined by Magiorakos et al. [3]) in the MRSN repository, 97 A. baumannii genomes of ST19 were identified.These were derived from isolates cultured from 97 patients between 2003 and 2023.The isolates originated from three distinct sources: (i) wounded US service members evacuated from Iraq and/or Afghanistan to military hospitals in Germany and/ or US, between 2003 and 2009, (ii) patients from six hospitals in Georgia between 2011 and 2022 and, (iii) patients hospitalised in Ukraine between 2014 and 2023; more details can be found in the Supplementary Table S1.
Antibiotic susceptibility tests were performed in the MRSN College of American Pathologists accredited clinical laboratory, as previously described [4], and interpreted using the Clinical and Laboratory Standards Institute 2023 guidelines [5].They revealed that just two of the 63 isolates (3%) collected between 2003 and 2018 were each non-susceptible to both imipenem and meropenem, while 32 of the 34 isolates collected since 2019 were non-susceptible to these two carbapenems, as detailed in Supplementary Table S1.
Finally, all non-redundant ST19 genomes available on GenBank (n = 11) were analysed: only two of six collected before 2019 carried a carbapenemase gene (one from Egypt with bla OXA-23 and one from Russia with bla OXA-72 ) while four of five isolates collected since 2019 carried bla OXA-23 (n = 2 from Egypt) or bla OXA- 72 (n = 2 from Russia) (Figure 1).

Genetic context of acquired carbapenemases in Acinetobacter baumannii ST19
Long-read sequencing was performed on seven representative isolates from SL1, -2 and -3 (Table ), as b Data were retrieved from the Multidrug-Resistant Organism Repository and Surveillance Network for 97 isolates and from GenBank for the remaining 11 strains.
previously described [7].Based on rep typing [8], circularised plasmids belonged to two groups, R3 and RP, except for three cryptic plasmids that carried no resistance genes (Table ).
For completeness, plasmid rep types were also identified for all the remaining ST19 isolates using Illumina short reads and mapped onto the phylogeny (Figure 2).The three carbapenemase genes, bla OXA- 23, bla OXA-72 and bla NDM-5 found in the three SLs appear to be independent acquisitions.For SL1, bla OXA- 72 was plasmid-bound (R3-type) and no significant homologies to composite transposons in the TnCentral database [9] were identified (Figure 3A).Concerning SL2, bla OXA-23 was chromosomal and carried by transposon Tn2008 [10] (Figure 3B).When present in SL2 isolates, bla NDM-5 was also chromosomal and carried by a 3.4 kb fragment of a truncated Tn125, inserted within ISAba1.In contrast, when present in SL2 isolates, bla OXA-72 was on a plasmid (R3-type) (Table and Figure 3).Of note, R3 plasmids from SL1 and SL2 isolates varied in length from 8.5 to 19.6 kb and an alignment confirmed that each lineage had distinct plasmid backbones despite an identical 7.0 kb insert harbouring bla OXA-72 (Figure 3A) .Finally, for the representative SL3 isolate, two identical copies of bla OXA-23 carried on resistance island AbaR4 were identified: one flanked by a 5 bp (5′-AAGGG-3′) target site duplication (TSD) within a RP-type plasmid and the second inserted within the chromosome (Figure 3).Interestingly, in this SL, and unlike a previous report [11], AbaR4 did not replace the ancestral AbaR3 island which remained intact within gene comM.Instead, AbaR4 was located ca 30 kb downstream, flanked by a 5′-AACTT-3′ TSD within chromosomal gene mutT.

Discussion
Multidrug-resistant A. baumannii has emerged as a leading cause of nosocomial infections worldwide [12].Of particular concern is the high rate of resistance to carbapenem antibiotics (> 50% in 21 European countries in 2020-2022 [13]), that makes treatment difficult [14].Carbapenem resistance in A. baumannii is generally attributed to either mutation and overexpression of the  intrinsic OXA-51-like carbapenemase or the acquisition of more potent carbapenemases (most often OXA-23 and OXA-24/40-like enzymes) via horizontal gene transfer [15].
The acquisition of carbapenemases, via horizontal gene transfer, is well characterised in ST1 and ST2, the preeminent lineages identified by multilocus sequence typing within global clones, GC1 and GC2, respectively [12,16].Nevertheless, GC complexes also include other lineages.One of these is ST19, a single locus variant of the ST1/GC1 clone, that has scarcely been reported globally [6,[17][18][19][20].At the time of the current study, we could find only 11 non-redundant genomes for A. baumannii ST19 in the GenBank database and, to the best of our knowledge, just five published reports of studies relating to this ST [6,[17][18][19][20].The first of these articles, published in 2015, described the detection of four ST19 isolates from hospital surfaces in Algeria, carrying bla NDM-5 [17].The second concerned a blood culture ST19 isolate in 2017 from a patient in Georgia, which harboured bla OXA-23 [20].The third in 2019, described ST19 isolates from an intensive care unit (ICU) in Italy [18].The fourth concerned three isolates in 2017−2019 from a neurological ICU in Moscow, Russia, all harbouring bla OXA-72 (an OXA-24/40 variant) [19] and the fifth was an article on three ST19 isolates from 2020 in Egypt and carrying bla OXA-23 on a chromosomally inserted Tn2006 transposon [6].
Amid 5,436 A. baumannii strains from 2003−2023 available in the MRSN repository, we found 97 of ST19.Among these ST19 strains, the proportions with carbapenem resistance appeared to be less (2/63) before 2019 than after (32/34) and, similarly, among 11 non-redundant ST19 genome sequences in GenBank, two of six from pre-2019 carried a carbapenemase gene while this was four of five post-2019.This could suggest that carbapenem-resistant A. baumannii ST19 are emerging or alternatively that these have previously been under-reported.Routine sampling accompanied by field epidemiology would be needed to fully understand their prevalence.Core genome SNPbased phylogenetic analysis of all 97 MRSN and 11 GenBank ST19 sequences revealed that, besides three monophyletic isolates from Egypt, carbapenemaseproducing isolates grouped into three SLs, including SL1, -2 and 3.The MRSN ST19 isolates that had been collected from 2019 onwards and that were nonsusceptible to carbapenems harboured either bla OXA- 23 , bla OXA-72 , and/or bla NDM-5 genes and were from Georgia and Ukraine.These isolates were found among the three SLs characterised in the current study.
A few limitations to this study can be noted.First, while the MRSN collected MDR A. baumannii from more than 20 countries on five continents [4], the presence of ST19 in other regions of the world remains to be investigated.Further, the volume and geographical origin of isolates sampled by the MRSN is changing through time.Hence, representative, and regular sampling and typing would be needed to better understand the prevalence of carbapenemase-producing ST19 A. baumannii globally.Finally, clonal isolates were not epidemiologically investigated.SL3, for instance, represents five highly genetically related isolates (7-42 SNPs) carrying bla OXA- 23 recovered from patients hospitalised in Ukraine in the same year (2023).It cannot be ruled out that isolates in SL3 represent an outbreak related to a single hospital.This could have inflated the numbers of ST19 isolates bearing carbapenemase genes, in particular for those collected after 2019.

Conclusion
While it is not possible to assess if incidence of carbapenemase-producing ST19 A. baumannii has recently increased in certain parts of the world, or if such strains have so far been under-detected, this report sheds more light on ST19, a ST barely described in the literature, yet circulating in several countries.The finding of multiple SLs of carbapenemase-producing ST19 A. baumannii from Georgia and Ukraine is concerning, as this might be exacerbated by the Russian invasion of Ukraine, which is causing disruption in infection prevention and control and movements of trauma patients.Learning from past protracted A. baumannii outbreaks occurring in US hospitals during military operations in Iraq and Afghanistan [23], it is imperative that programmes to detect carbapenemase-producing A. baumannii in facilities treating war wounded people be implemented and/or reinforced.Finally, this report further emphasises the key role of global epidemiological and genomic surveillance programmes to address antimicrobial resistance.Information (NCBI) database under the BioProject number PRJNA1101874.

Table
Identification of plasmids and location of carbapenemase genes in representative ST19 Acinetobacter baumannii isolates with complete genomes, 2011−2023 (n = 7 isolates) When present, the plasmid size is indicated for RP-and R3-types as well as for cryptic plasmids (others).b Carbapenemase gene inferred location on the chromosome (Chr) or plasmids (RP-and R3-types).When applicable, the carriage by transposons (Tn2008 or truncated ΔTn125) or resistance island (AbaR4) is indicated. a